Structural insight into the antibiotic action of telithromycin against resistant mutants

The crystal structure of the ketolide telithromycin bound to the Deinococcus radiodurans large ribosomal subunit shows that telithromycin blocks the ribosomal exit tunnel and interacts with domains II and V of the 23S RNA. Comparisons to other clinically relevant macrolides provided structural insights into its enhanced activity against macrolide-resistant strains.     

Structural insight into the altered substrate specificity of human cytochrome P450 2A6 mutants

Human P450 2A6 displays a small active site that is well adapted for the oxidation of small planar substrates. Mutagenesis of CYP2A6 resulted in an increased catalytic efficiency for indole biotransformation to pigments and conferred a capacity to oxidize substituted indoles (Wu, Z.-L., Podust, L.M., Guengerich, F.P. J. Biol. Chem. 49 (2005) 41090-41100.). Here, we describe the structural basis that underlies the altered metabolic profile of three mutant enzymes, P450 2A6 N297Q, L240C/N297Q and N297Q/I300V. The Asn297 substitution abolishes a potential hydrogen bonding interaction with substrates in the active site, and replaces a structural water molecule between the helix B'-C region and helix I while maintaining structural hydrogen bonding interactions. The structures of the P450 2A6 N297Q/L240C and N297Q/I300V mutants provide clues as to how the protein can adapt to fit the larger substituted indoles in the active site, and enable a comparison with other P450 family 2 enzymes for which the residue at the equivalent position was seen to function in isozyme specificity, structural integrity and protein flexibility.     

Structural insight into mechanism and diverse substrate selection strategy of L-ribulokinase

The araBAD operon encodes three different enzymes required for catabolism of L-arabinose, which is one of the most abundant monosaccharides in nature. L-ribulokinase, encoded by the araB gene, catalyzes conversion of L-ribulose to L-ribulose-5-phosphate, the second step in the catabolic pathway. Unlike other kinases, ribulokinase exhibits diversity in substrate selectivity and catalyzes phosphorylation of all four 2-ketopentose sugars with comparable k(cat) values. To understand ribulokinase recognition and phosphorylation of a diverse set of substrates, we have determined the X-ray structure of ribulokinase from Bacillus halodurans bound to L-ribulose and investigated its substrate and ATP co-factor binding properties. The polypeptide chain is folded into two domains, one small and the other large, with a deep cleft in between. By analogy with related sugar kinases, we identified (447)GGLPQK(452) as the ATP-binding motif within the smaller domain. L-ribulose binds in the cleft between the two domains via hydrogen bonds with the side chains of highly conserved Trp126, Lys208, Asp274, and Glu329 and the main chain nitrogen of Ala96. The interaction of L-ribulokinase with L-ribulose reveals versatile structural features that help explain recognition of various 2-ketopentose substrates and competitive inhibition by L-erythrulose. Comparison of our structure to that of the structures of other sugar kinases revealed conformational variations that suggest domain-domain closure movements are responsible for establishing the observed active site environment.     

Structural insight into poly(A) binding and catalytic mechanism of human PARN

Poly(A)-specific ribonuclease (PARN) is a processive, poly(A)-specific 3' exoribonuclease. The crystal structure of C-terminal truncated human PARN determined in two states (free and RNA-bound forms) reveals that PARNn is folded into two domains, an R3H domain and a nuclease domain similar to those of Pop2p and epsilon186. The high similarity of the active site structures of PARNn and epsilon186 suggests that they may have a similar catalytic mechanism. PARNn forms a tight homodimer, with the R3H domain of one subunit partially enclosing the active site of the other subunit and poly(A) bound in a deep cavity of its nuclease domain in a sequence-nonspecific manner. The R3H domain and, possibly, the cap-binding domain are involved in poly(A) binding but these domains alone do not appear to contribute to poly(A) specificity. Mutations disrupting dimerization abolish both the enzymatic and RNA-binding activities, suggesting that the PARN dimer is a structural and functional unit. The cap-binding domain may act in concert with the R3H domain to amplify the processivity of PARN.     

Structural insight into the rotational switching mechanism of the bacterial flagellar motor

The bacterial flagellar motor can rotate either clockwise (CW) or counterclockwise (CCW). Three flagellar proteins, FliG, FliM, and FliN, are required for rapid switching between the CW and CCW directions. Switching is achieved by a conformational change in FliG induced by the binding of a chemotaxis signaling protein, phospho-CheY, to FliM and FliN. FliG consists of three domains, FliG(N), FliG(M), and FliG(C), and forms a ring on the cytoplasmic face of the MS ring of the flagellar basal body. Crystal structures have been reported for the FliG(MC) domains of Thermotoga maritima, which consist of the FliG(M) and FliG(C) domains and a helix E that connects these two domains, and full-length FliG of Aquifex aeolicus. However, the basis for the switching mechanism is based only on previously obtained genetic data and is hence rather indirect. We characterized a CW-biased mutant (fliG(ΔPAA)) of Salmonella enterica by direct observation of rotation of a single motor at high temporal and spatial resolution. We also determined the crystal structure of the FliG(MC) domains of an equivalent deletion mutant variant of T. maritima (fliG(ΔPEV)). The FliG(ΔPAA) motor produced torque at wild-type levels under a wide range of external load conditions. The wild-type motors rotated exclusively in the CCW direction under our experimental conditions, whereas the mutant motors rotated only in the CW direction. This result suggests that wild-type FliG is more stable in the CCW state than in the CW state, whereas FliG(ΔPAA) is more stable in the CW state than in the CCW state. The structure of the TM-FliG(MC)(ΔPEV) revealed that extremely CW-biased rotation was caused by a conformational change in helix E. Although the arrangement of FliG(C) relative to FliG(M) in a single molecule was different among the three crystals, a conserved FliG(M)-FliG(C) unit was observed in all three of them. We suggest that the conserved FliG(M)-FliG(C) unit is the basic functional element in the rotor ring and that the PAA deletion induces a conformational change in a hinge-loop between FliG(M) and helix E to achieve the CW state of the FliG ring. We also propose a novel model for the arrangement of FliG subunits within the motor. The model is in agreement with the previous mutational and cross-linking experiments and explains the cooperative switching mechanism of the flagellar motor.     

Structural insight into the mechanism of streptozotocin inhibition of O-GlcNAcase

Despite decades of its use in diabetes research, the mechanism of cytotoxicity of streptozotocin (STZ) toward pancreatic β-islet cells has remained a topic of discussion. Although STZ toxicity is likely a function of its capacity to promote DNA alkylation, it has been proposed that STZ induces pancreatic β-cell death through O-GlcNAcase inhibition. In this report, we explore the binding mode of STZ to a close homolog of human O-GlcNAcase, BtGH84 from Bacteroides thetaiotaomicron. Our results show that STZ binds in the enzyme active site in its intact form, without the formation of a covalent adduct, consistent with solution studies on BtGH84 and human O-GlcNAcase, as well as with structural work on a homolog from Clostridium perfringens. The active site of the BtGH84 is considerably deformed upon STZ binding and as a result the catalytic machinery is expelled from the binding cavity.     

Structural insight into the mechanism of substrate specificity of aedes kynurenine aminotransferase

Aedes aegypti kynurenine aminotransferase (AeKAT) is a multifunctional aminotransferase. It catalyzes the transamination of a number of amino acids and uses many biologically relevant alpha-keto acids as amino group acceptors. AeKAT also is a cysteine S-conjugate beta-lyase. The most important function of AeKAT is the biosynthesis of kynurenic acid, a natural antagonist of NMDA and alpha7-nicotinic acetylcholine receptors. Here, we report the crystal structures of AeKAT in complex with its best amino acid substrates, glutamine and cysteine. Glutamine is found in both subunits of the biological dimer, and cysteine is found in one of the two subunits. Both substrates form external aldemines with pyridoxal 5-phosphate in the structures. This is the first instance in which one pyridoxal 5-phosphate enzyme has been crystallized with cysteine or glutamine forming external aldimine complexes, cysteinyl aldimine and glutaminyl aldimine. All the units with substrate are in the closed conformation form, and the unit without substrate is in the open form, which suggests that the binding of substrate induces the conformation change of AeKAT. By comparing the active site residues of the AeKAT-cysteine structure with those of the human KAT I-phenylalanine structure, we determined that Tyr286 in AeKAT is changed to Phe278 in human KAT I, which may explain why AeKAT transaminates hydrophilic amino acids more efficiently than human KAT I does.     

Structural insight into the mechanism of double-stranded RNA processing by ribonuclease III

Members of the ribonuclease III (RNase III) family are double-stranded RNA (dsRNA) specific endoribonucleases characterized by a signature motif in their active centers and a two-base 3' overhang in their products. While Dicer, which produces small interfering RNAs, is currently the focus of intense interest, the structurally simpler bacterial RNase III serves as a paradigm for the entire family. Here, we present the crystal structure of an RNase III-product complex, the first catalytic complex observed for the family. A 7 residue linker within the protein facilitates induced fit in protein-RNA recognition. A pattern of protein-RNA interactions, defined by four RNA binding motifs in RNase III and three protein-interacting boxes in dsRNA, is responsible for substrate specificity, while conserved amino acid residues and divalent cations are responsible for scissile-bond cleavage. The structure reveals a wealth of information about the mechanism of RNA hydrolysis that can be extrapolated to other RNase III family members.     

Structural characterization of the Myxococcus xanthus encapsulin and ferritin-like cargo system gives insight into its iron storage mechanism

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Functional and physical competition between phospholamban and its mutants provides insight into the molecular mechanism of gene therapy for heart failure

We have used functional co-reconstitution of purified sarcoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA) with phospholamban (PLB), its inhibitor in the heart, to test the hypothesis that loss-of-function (LOF) PLB mutants (PLB_M) can compete with wild-type PLB (PLB_W) to relieve SERCA inhibition. Co-reconstitution at varying PLB-to-SERCA ratios was conducted using synthetic PLB_W, gain-of-function mutant I40A, or LOF mutants S16E (phosphorylation mimic) or L31A. Inhibitory potency was defined as the fractional increase in K_Ca, measured from the Ca²⁺-dependence of ATPase activity. At saturating PLB, the inhibitory potency of I40A was about three times that of PLB_W, while the potency of each of the LOF PLB_M was about one third that of PLB_W. However, there was no significant variation in the apparent SERCA affinity for these four PLB variants. When SERCA was co-reconstituted with mixtures of PLB_W and LOF PLB_M, inhibitory potency was reduced relative to that of PLB_W alone. Furthermore, FRET between donor-labeled SERCA and acceptor-labeled PLB_W was decreased by both (unlabeled) LOF PLB_M. These results show that LOF PLB_M can compete both physically and functionally with PLB_W, provide a rational explanation for the partial success of S16E-based gene therapy in animal models of heart failure, and establish a powerful platform for designing and testing more effective PLB_M targeted for gene therapy of heart failure in humans.     

Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis

Alkaline exonuclease and single-strand DNA (ssDNA) annealing proteins (SSAPs) are key components of DNA recombination and repair systems within many prokaryotes, bacteriophages and virus-like genetic elements. The recently sequenced β-proteobacterium Laribacter hongkongensis (strain HLHK9) encodes putative homologs of alkaline exonuclease (LHK-Exo) and SSAP (LHK-Bet) proteins on its 3.17 Mb genome. Here, we report the biophysical, biochemical and structural characterization of recombinant LHK-Exo protein. LHK-Exo digests linear double-stranded DNA molecules from their 5′-termini in a highly processive manner. Exonuclease activities are optimum at pH 8.2 and essentially require Mg²⁺ or Mn²⁺ ions. 5′-phosphorylated DNA substrates are preferred over dephosphorylated ones. The crystal structure of LHK-Exo was resolved to 1.9 Å, revealing a ‘doughnut-shaped’ toroidal trimeric arrangement with a central tapered channel, analogous to that of λ-exonuclease (Exo) from bacteriophage-λ. Active sites containing two bound Mg²⁺ ions on each of the three monomers were located in clefts exposed to this central channel. Crystal structures of LHK-Exo in complex with dAMP and ssDNA were determined to elucidate the structural basis for substrate recognition and binding. Through structure-guided mutational analysis, we discuss the roles played by various active site residues. A conserved two metal ion catalytic mechanism is proposed for this class of alkaline exonucleases.     

Structural insight into the binding mechanism of ATP to EGFR and L858R,and T790M and L858R/T790 mutants

Abstract

The L858R mutation in EGFR is particularly responsive to small tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib. This efficacy decreases due to drug resistance conferred by a second mutation, T790M, which subsequently produces a double mutant, L858R/T790M. Although this resistance was initially attributed to steric blocking by the T790M mutation, experimental studies have demonstrated that differences in the binding affinities of TKIs to T790M and L858R/T790M mutants are more a result of the increased sensitivity of these mutants to ATP than to a decrease in the affinity to TKIs. Regrettably, detailed information at the atomic level on the origins of the increased binding affinity of mutants for ATP is lacking. In this study, we have combined structural data and molecular dynamics simulations with the MMGBSA approach to determine how the L858R, T790M and L858R/T790 mutations impact the binding mechanism of ATP with respect to wild-type EGFR. Structural and energetic analyses provided novel information that helps to explain the increased affinity of ATP to T790M and L858R/T790 mutants with respect to L858R and wild-type systems. In addition, it was observed that dimerization of the wild-type and mutant systems exerts dissimilar effects on the ATP binding affinity characteristic of negative cooperativity.

Communicated by Ramaswamy H. Sarma     

A novel insight into the mechanism of mammalian selenoprotein synthesis

The amino acid selenocysteine is encoded by UGA, usually a stop codon, thus requiring a specialized machinery to enable its incorporation into selenoproteins. The machinery comprises the tRNA^Sec, a 3′-UTR mRNA stem–loop termed SElenoCysteine Insertion Sequence (SECIS), which is mandatory for recoding UGA as a Sec codon, the SECIS Binding Protein 2 (SBP2), and other proteins. Little is known about the molecular mechanism and, in particular, when, where, and how the SECIS and SBP2 contact the ribosome. Previous work by others used the isolated SECIS RNA to address this question. Here, we developed a novel approach using instead engineered minimal selenoprotein mRNAs containing SECIS elements derivatized with photoreactive groups. By cross-linking experiments in rabbit reticulocyte lysate, new information could be gained about the SBP2 and SECIS contacts with components of the translation machinery at various translation steps. In particular, we found that SBP2 was bound only to the SECIS in 48S pre-initiation and 80S pretranslocation complexes. In the complex where the Sec-tRNA^Sec was accommodated to the A site but transpeptidation was blocked, SBP2 bound the ribosome and possibly the SECIS element as well, and the SECIS had flexible contacts with the 60S ribosomal subunit involving several ribosomal proteins. Altogether, our findings led to broadening our understanding about the unique mechanism of selenocysteine incorporation in mammals.     

Structural insight into the ESCRT-I/-II link and its role in MVB trafficking

ESCRT (endosomal sorting complex required for transport) complexes orchestrate efficient sorting of ubiquitinated transmembrane receptors to lysosomes via multivesicular bodies (MVBs). Yeast ESCRT-I and ESCRT-II interact directly in vitro; however, this association is not detected in yeast cytosol. To gain understanding of the molecular mechanisms of this link, we have characterised the ESCRT-I/-II supercomplex and determined the crystal structure of its interface. The link is formed by the vacuolar protein sorting (Vps)28 C-terminus (ESCRT-I) binding with nanomolar affinity to the Vps36-NZF-N zinc-finger domain (ESCRT-II). A hydrophobic patch on the Vps28-CT four-helix bundle contacts the hydrophobic knuckles of Vps36-NZF-N. Mutation of the ESCRT-I/-II link results in a cargo-sorting defect in yeast. Interestingly, the two Vps36 NZF domains, NZF-N and NZF-C, despite having the same core fold, use distinct surfaces to bind ESCRT-I or ubiquitinated cargo. We also show that a new component of ESCRT-I, Mvb12 (YGR206W), engages ESCRT-I directly with nanomolar affinity to form a 1:1:1:1 heterotetramer. Mvb12 does not affect the affinity of ESCRT-I for ESCRT-II in vitro. Our data suggest a complex regulatory mechanism for the ESCRT-I/-II link in yeast.     

Structural insight into the bifunctional mechanism of the glycogen-debranching enzyme TreX from the archaeon Sulfolobus solfataricus

TreX is an archaeal glycogen-debranching enzyme that exists in two oligomeric states in solution, as a dimer and tetramer. Unlike its homologs, TreX from Sulfolobus solfataricus shows dual activities for alpha-1,4-transferase and alpha-1,6-glucosidase. To understand this bifunctional mechanism, we determined the crystal structure of TreX in complex with an acarbose ligand. The acarbose intermediate was covalently bound to Asp363, occupying subsites -1 to -3. Although generally similar to the monomeric structure of isoamylase, TreX exhibits two different active-site configurations depending on its oligomeric state. The N terminus of one subunit is located at the active site of the other molecule, resulting in a reshaping of the active site in the tetramer. This is accompanied by a large shift in the "flexible loop" (amino acids 399-416), creating connected holes inside the tetramer. Mutations in the N-terminal region result in a sharp increase in alpha-1,4-transferase activity and a reduced level of alpha-1,6-glucosidase activity. On the basis of geometrical analysis of the active site and mutational study, we suggest that the structural lid (acids 99-97) at the active site generated by the tetramerization is closely associated with the bifunctionality and in particular with the alpha-1,4-transferase activity. These results provide a structural basis for the modulation of activities upon TreX oligomerization that may represent a common mode of action for other glycogen-debranching enzymes in higher organisms.     

Structural and functional analysis of the HIV gp41 core containing an Ile573 to Thr substitution: implications for membrane fusion

The envelope glycoprotein of HIV-1 consists of the surface subunit gp120 and the transmembrane subunit gp41. Binding of gp120 to target cell receptors induces a conformational change in gp41, which then mediates the fusion of viral and cellular membranes. A buried isoleucine (Ile573) in a central trimeric coiled coil within the fusion-active gp41 ectodomain core is thought to favor this conformational activation. The role of Ile573 in determining the structure and function of the gp120-gp41 complex was investigated by mutating this residue to threonine, a nonconservative substitution in HIV-1 that occurs naturally in SIV. While the introduction of Thr573 markedly destabilized the gp41 core, the three-dimensional structure of the mutant trimer of hairpins was very similar to that of the wild-type molecule. A new hydrogen-bonding interaction between the buried Thr573 and Thr569 residues appears to allow formation of the trimer-of-hairpins structure at physiological temperature. The mutant envelope glycoprotein expressed in 293T cells and incorporated within pseudotyped virions displayed only a moderate reduction in syncytium-inducing capacity and virus infectivity, respectively. Our results demonstrate that the proper folding of the gp41 core underlies the membrane fusion properties of the gp120-gp41 complex. An understanding of the gp41 activation process may suggest novel strategies for vaccine and antiviral drug development.     

Structural and biochemical insight into the mechanism of dual CpG site binding and methylation by the DNMT3A DNA methyltransferase

DNMT3A/3L heterotetramers contain two active centers binding CpG sites at 12 bp distance, however their interaction with DNA not containing this feature is unclear. Using randomized substrates, we observed preferential co-methylation of CpG sites with 6, 9 and 12 bp spacing by DNMT3A and DNMT3A/3L. Co-methylation was favored by AT bases between the 12 bp spaced CpG sites consistent with their increased bending flexibility. SFM analyses of DNMT3A/3L complexes bound to CpG sites with 12 bp spacing revealed either single heterotetramers inducing 40° DNA bending as observed in the X-ray structure, or two heterotetramers bound side-by-side to the DNA yielding 80° bending. SFM data of DNMT3A/3L bound to CpG sites spaced by 6 and 9 bp revealed binding of two heterotetramers and 100° DNA bending. Modeling showed that for 6 bp distance between CpG sites, two DNMT3A/3L heterotetramers could bind side-by-side on the DNA similarly as for 12 bp distance, but with each CpG bound by a different heterotetramer. For 9 bp spacing our model invokes a tetramer swap of the bound DNA. These additional DNA interaction modes explain how DNMT3A and DNMT3A/3L overcome their structural preference for CpG sites with 12 bp spacing during the methylation of natural DNA.     

基于代谢组的厌氧氨氧化菌群对温度的响应机制

【背景】在环境工程领域中,大多数耐冷菌从活性污泥中分离得到。了解活性污泥菌群对低温的响应有助于耐冷菌的驯化培养。【目的】以厌氧氨氧化污泥菌群作为研究对象,研究温度对厌氧氨氧化菌群代谢通路与代谢产物的影响,以期初步阐释厌氧氨氧化菌群低温响应机理。【方法】在25°C与35°C条件下驯化培养厌氧氨氧化污泥,研究温度对反应器脱氮效能、菌群活性与生长以及群落结构的影响,通过代谢组学比较两个温度下厌氧氨氧化菌群代谢物丰度以及代谢通路活性。【结果】虽然低温导致厌氧氨氧化菌群CO_2固定、TCA循环与丙酮酸代谢的下调,进而导致氮去除活性以及增长速率显著下降,但菌群RNA合成水平、腐胺与信号分子合成上调,从而通过转录调控、调控膜脂组成与改变膜结构的方式,调控菌群代谢以适应低温环境。【结论】从分子机理的角度探究了厌氧氨氧化污泥菌群适应低温环境的生理机制,首次阐释了腐胺与信号分子在污泥菌群适应低温过程中的重要作用。     

Crystal structure of Escherichia coli ketopantoate reductase at 1.7 A resolution and insight into the enzyme mechanism.

Ketopantoate reductase (KPR, EC 1.1.1.169) catalyzes the NADPH-dependent reduction of ketopantoate to pantoate on the pantothenate (vitamin B(5)) biosynthetic pathway. The Escherichia coli panE gene encoding KPR was cloned and expressed at high levels as the native and selenomethionine-substituted (SeMet) proteins. Both native and SeMet recombinant proteins were purified by three chromatographic steps, to yield pure proteins. The wild-type enzyme was found to have a K(M)(NADPH) of 20 microM, a K(M)(ketopantoate) of 60 microM, and a k(cat) of 40 s(-1). Regular prismatic KPR crystals were prepared using the hanging drop technique. They belonged to the tetragonal space group P4(2)2(1)2, with cell parameters: a = b = 103.7 A and c = 55.7 A, accommodating one enzyme molecule per asymmetric unit. The structure of KPR was determined by the multiwavelength anomalous dispersion method using the SeMet protein, for which data were collected to 2.3 A resolution. The native data were collected to 1.7 A resolution and used to refine the final structure. The secondary structure comprises 12 alpha-helices, three 3(10)-helices, and 11 beta-strands. The enzyme is monomeric and has two domains separated by a cleft. The N-terminal domain has an alphabeta-fold of the Rossmann type. The C-terminal domain (residues 170-291) is composed of eight alpha-helices. KPR is shown to be a member of the 6-phosphogluconate dehydrogenase C-terminal domain-like superfamily. A model for the ternary enzyme-NADPH-ketopantoate ternary complex provides a rationale for kinetic data reported for specific site-directed mutants.